Imaging of mammalian tissues has been used extensively to obtain information on the internal structures as well as on the biodistribution of molecules. This information can of course be utilized for diagnosis purposes. Several techniques based on different physical principles are currently available to obtain images that encompass a broad range of spatio-temporal resolution. Such techniques include Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), single-photon emission computed tomography (SPECT), X-ray, ultrasound and, now emerging, optical imaging.
In optical imaging, three approaches have been used to generate the optical data necessary to reconstruct images of volume of interest (VOI). The continuous wave (CW) technique uses a continuous wave light source and allows the measurement of light attenuation. The time domain (TD) technique involves injecting a pulse of light of short duration into the VOI and detecting the light as a function of time as it exits the VOI. Finally, the frequency domain (FD) technique relies on frequency modulation of a light source and analysis of the phase and amplitude of the resulting optical signal as it exits the VOI. Together, time domain and frequency domain may be referred to as “time-resolved” (TR).
Characterization of the VOI in optical imaging relies on a determination of the absorption and scattering characteristics of the different regions of the VOI. Starting with assumptions for absorption and scattering values, measurements are then taken and used to modify these assumptions to improve the image data. Light that is injected into the VOI at one point is detected as it exits at another point, and the detected signal provides information regarding the region through which the light passed. Attenuation of the light transmitted through the VOI due to absorption and scattering is quantifiable at the detector. Moreover, the TD technique allows the generation of a time point spread function (TPSF), which provides additional information regarding the extent to which the attenuation is due to absorption or due to scattering. This allows the decoupling of the two primary detected optical parameters of interest, the absorption coefficient μa and the reduced scattering coefficient μs′. By sampling the VOI using a number of different source and detector positions, more detailed spatial information regarding the structure of the VOI is obtained, allowing a three-dimensional image to be constructed.
Fluorescence diffuse optical tomography (FDOT) systems obtain three-dimensional images showing the location and number density of fluorescent molecules embedded in a biological medium. Typically, this is achieved using a large set of surface measurements combined to a photon propagation model. For highly scattering media, (μs′>10μa), photon propagation is well approximated by solutions to the diffusion equation. A further simplification that is often made consists in using analytical solutions to the diffusion equation for forward model building. In this case, an assumption is made that the absorption and scattering properties of the medium are constant throughout. This approximation is convenient when considering commercial devices because it allows fluorescence image reconstructions to be performed in a relatively short processing time. For in vivo imaging however, the optical property heterogeneities associated with the non-trivial anatomy of the animal cannot simply be neglected. To that effect, more sophisticated (and time-consuming) methods exist where photon propagation is actually computed using the a priori information related to organs and tissues (spatial distribution and optical properties). Aside from the use of a priori structural information, proposals have been made in the past to help minimize the impact of optical heterogeneities on fluorescence data. For example, the Born normalization scheme for tomography data sets has been experimentally shown to significantly improve the quality of reconstructed fluorescence images.
For a heterogeneous sample, such as a small animal, the variations in composition of the sample from one location to another complicate the problem of acquiring an accurate image of the VOI. In the absence of a priori structural and anatomical information obtained through another modality such as microCT, PET, SPECT or MRI systems, conventional FDOT systems typically make the assumption of constant absorption and scattering properties throughout the medium, as mentioned above. However, such an assumption is crude, and is particularly ineffective when performing imaging in vivo, and the alternative of using information gathered from another modality may be unattractive as it significantly complicates the imaging process.